Saponins can be found in more than one hundred plant families and in some marine animals. However, chemical investigation of soybean (Glycine max L. Merrill) saponins has begun only as recently as the 1970s. Here we focus on the chemical structure, the content, and biological activity of soybean saponins in current studies. Especially, we focus on 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) conjugated saponins and define the chemical structure of this as a natural precursor of group B and E saponins.
Photocatalytic production of the electron (e-) and positive hole (h+) in an aqueous suspension of TiO2 (anatase form) under illumination by near-UV light (295-390 nm) generated the superoxide (O2-) and hydroxyl radical (•OH), which both proceeded linearly with reaction time, while H2O2 accumulated non-linearly. Under anaerobic conditions (introduced Ar gas), the yields of three active species of oxygen were decreased to 10-20% of those detected in the air-saturated reaction. The electron spin resonance (ESR) signal characteristics of •OH were obtained when a spin trap of 5,5-dimthyl-1-pyrroline-N-oxide (DMPO) was included in the illuminating mixture. The intensity of the ESR signal was increased by Cu/Zn superoxide dismutase, and decreased under anaerobic conditions, amounting to only 20% of the intensity detected in the aerobic reaction. The addition of H2O2 to the reaction mixture resulted in about an 8-fold increase of •OH production in the anaerobic reaction, but only about 1.5-fold in the aerobic reaction, indicating that e- generated by the photocatalytic reaction reduced H2O2 to produce •OH plus OH-. On the other hand, D2O lowered the yield of •OH generation to 18% under air and 40% under Ar conditions, indicating the oxidation of H2O by h+. The addition of Fe(III)-EDTA as an electron acceptor effectively increased •OH generation, 2.3-fold in the aerobic reaction and 8.4-fold in the anaerobic reaction, the yield in the latter exceeding that in the air-saturated reaction.
A convenient and quantitative bioassay method for evaluating the efficiency of plant transformation by Agrobacterium tumefaciens is important to search plant transformation inhibitors, possible biochemical probes for study on its mechanism. Our previously reported method, in which the plant transformation had been detected by the expression of β-glucuronidase in transformed plants, was improved. The difference between the previous and the improved methods is the use of suspension-cultured cells of Ageratum conyzoides as the host plant instead of Nicotiana tabacum BY-2; this alteration of the host enabled us to measure the β-glucuronidase activity in plant cells not only fluorometrically but also colorimetrically. The enzyme activity expressed in the cells of A. conyzoides was nearly 100 times higher than that of N. tabacum BY-2, and was enough for detection by colorimetric measurement. The method, therefore, is useful for a convenient determination of inhibitory activity against plant transformation.
Optically active 1,4-thiazane-3-carboxylic acid [TCA] was synthesized from cysteine via optical resolution by preferential crystallization. The intermediate (RS)-2-amino-3-[(2-chloroethyl)sulfanyl]propanoic acid hydrochlo-ride [(RS)-ACS•HCl] was found to exist as a conglomerate based on its melting point, solubility and IR spectrum. (RS)-ACS•HCl was optically resolved by preferential crystallization to yield (R)- and (S)-ACS•HCl. (R)- and (S)-ACS•HCl thus obtained were recrystallized from a mixture of hydrochloric acid and 2-propanol, taking account of the solubility of (RS)-ACS•HCl, efficiently yielding both enantiomers in optically pure forms. (R)- and (S)-TCA were then respectively synthesized by the cyclization of (R)- and (S)-ACS•HCl in ethanol in the presence of triethylamine.
A crucial enzyme in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which is responsible for the initial carbocycle formation of 2-deoxy-scyllo-inosose (1) from D-glucose-6-phosphate (G-6-P) (2). To get more insight into the mechanism and substrate specificity, deoxy-D-glucose-6-phosphates (deoxy-G-6-P) were chemically synthesized and subjected to the reaction with DOIS. The enzyme appeared to use 2-deoxy- and 3-deoxy-G-6-P as substrates, both of which were converted into the corresponding dideoxy-scyllo-inosose products, but 4-deoxy-G-6-P failed in cyclization by DOIS. These results clearly support the proposed reaction mechanism involving the initial oxidation at C-4 of the G-6-P substrate. Another implication is the potential use of DOIS for the preparation of useful dideoxyinososes.
Both enantiomers of the C7-C12 segment (3 and 4) of antitumor antibiotics epothilones (1and 2) were synthesized from methyl (R)- and (S)-3-hydroxy-2-methylpropionate (5 and 6) in five steps in a fair yield.
Enantiomerically pure (2S,4aS,8S)-(+)-4a,8-dimethyl-2,3,4,4a,5,6,7,8-octahydro-2-naphthalenol (3), a key intermediate in the synthesis of natural geosmin (1), was prepared by enzymatic kinetic resolution. When racemic 3 was submitted to lipase (PS-30)-catalyzed asymmetric acetylation, employing vinyl acetate as the acyl donor, requisite product (+)-3 with a high enantiomeric excess was attained as the remaining alcohol. Recrystallization resulted in an enantiomerically pure sample.
The structures of new antibacterial diterpenoids that had been isolated from Sarcodon scabrosus were established by chemical and spectral means to be sarcodonin L (2) and M (3), both having the cyathane skeleton. Other antibacterial compounds were identified to be allocyathin B2 (1), sarcodonin G (4) and sarcodonin A (5) by comparing their spectral data with those of authentic samples.
A novel cyclohexene compound (1), which is structurally related to theobroxide (2), was isolated from a culture filtrate of the fungus, Lasiodiplodia theobromae IFO 31059. The potato micro-tuber-inducing activity of this compound was observed at a concentration of 10-3 M in the medium, whereas theobroxide (2) showed its activity at 10-5 M.
Highly branched xyloglucan oligosaccharides were analyzed by the post-source decay (PSD) fragmentation method of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ratio of [M-Xyl]+ and [M-Gal]+ fragment ion intensities could be used to characterize the degree of Gal substitution at the non-reducing end, because the number of possible chemical species was directly related to their relative ion intensity. The intensity of the [M-Fuc]+ ion was predominantly strong in the fragment spectrum of fucosyl oligosaccharides as the first fragmentation, indicating the fucosyl linkage to be much weaker than the other glycosidic linkages in the MALDI-PSD fragmentation. Setting fragment ion [M-Fuc]+ to the pseudo precursor ion [MF]+, the second fragmentation ions were produced from [MF]+ in the drift region in PSD fragmentation of fucosyl oligosaccharides.
NMR analyses of striatol produced by suspension-cultured cells of liverwort, Ptycanthus striatus, in the presence of [2-13C]- and [4,4-2H2]-MVAs confirmed proton-catalyzed cyclization between the distal and central double bonds in FPP and a concerted series of 1,2-migrations of hydrogen and a methyl group, with subsequent elimination of a proton.
An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp. YY62 with 9% overall yield. Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SWXL gel-filtration and phenyl 5PW hydrophobic interaction columns). Enzymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO-hydrolyzing activity. The peak preparation showed a single band of 40 kDa on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase. The lipase showed maximum activity at pH 7.0 and 28°C. The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase.
We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombinant plasmid pLC1-hph, which contains the L. edodes transcriptional signals and an Escherichia coli hygromycin B phosphotransferase gene. Protoplasts of L. edodes were treated by the PEG transformation mixture containing 50 units of SalI, which cleaves pLC1-hph at a single site, yielding about 15 transformants per 2.5 μg of DNA. The conventional PEG transformation without SalI, however, yielded only 1.5 transformants per 25 μg of DNA. The optimal amount of SalI for increased transformation was 50 units. In the case of transformation with SphI, which cleaves the plasmid at one site, the optimal amount of the enzyme was 2.5 units. Southern blot analysis of the SphI-derived transformants suggested that 50% of the plasmid integrations were REMI events.
We have investigated whether niacin-related compounds act as inducers of apoptosis in HL-60 cells. In this study, we found that picolinic acid, dipicolinic acid, and isonicotinamide strongly induce apoptosis. After treatments with these compounds, apoptosis started within 4 h and was induced in about 50% of the cells within 8 h. These compounds induced apoptosis at 5-10 mM, but did not at 1 mM. An ICE-like protease inhibitor (Z-Asp-CH2-DCB) completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CHO) and a caspase-3 inhibitor (Ac-DEVD-CHO) did not block the apoptosis, suggesting that other caspases have the critical roles in the execution process of apoptosis induced by niacin-related compounds.
Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a crude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme showed maximal activity at about pH 10.9 for the deamination of L-alanine and at about pH 8.7 for the amination of pyruvate. The enzyme required NAD+ as a coenzyme. Analogs of NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide served as coenzymes. Initial-velocity and product inhibition studies suggested that the deamination of L-alanine proceeded through a sequential ordered binary-ternary mechanism. NAD+ bound first to the enzyme, followed by L-alanine, and the products were released in the order of ammonia, pyruvate, and NADH. The Km were 0.47 mM for L-alanine, 0.16 mM for NAD+, 0.22 mM for pyruvate, 0.067 mM for NADH, and 66.7 mM for ammonia. The Km for L-alanine was the smallest in the alanine dehydrogenases studied so far. The enzyme gene was cloned into Escherichia coli JM109 cells and the nucleotides were sequenced. The deduced amino acid sequence was very similar to that of the alanine dehydrogenase from Bacillus subtilis. However, the Enterobacter enzyme has no cysteine residue. In this respect, the Enterobacter enzyme is different from other alanine dehydrogenases.
A carbon catabolite repressor gene, creA, was isolated from the cellulolytic fungus Humicola grisea by using a portion of the Trichoderma reesei cre1 gene as a probe. The deduced amino acid sequence predicts a zinc finger protein of 419 amino acids in length, and its zinc finger regions show high similarity with those of Aspergillus CreAs, T. reesei Cre1, and Saccharomyces cerevisiae MIG1. Northern blot analysis showed that the H. grisea creA gene was highly transcribed when the mycelia were grown on glucose-containing media, but the transcription of the H. grisea endoglucanase 1 gene (egl1) and the exoglucanase 1 gene (exo1) were repressed under these conditions. Results of binding assays with the maltose-binding protein::CreA(1-166) fusion protein and the egl1 and the exo1 upstream regions showed that some 6-bp sites having an identical or similar sequence to the consensus sequence for CreA binding were protected from DNase I digestion.
Glycerol kinase (EC 220.127.116.11) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22°C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes (Biochemistry, 18:5698-5703, 1979).
A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.
The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNAArg, cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obatined after removal of heat denatured E. coli proteins.
A new thiamin glycoside, e.g., thiamin β-D-2-deoxy-2-acetamidoglucopyranoside was prepared by transglycosylation by β-N-acetylhexosaminidase from Aspergillus oryzae and characterized spectrally. Series of other fungal β-N-acetyl-hexosaminidases from A. awamori, A. tamari, A. terreus, and Penicillium oxalicum were shown to be able to synthesize the same compound in lower yields.
A method for rapidly identifying six of the most commonly found xyloglucan oligosaccharide units, XXXG, XLXG, XXLG, XLLG, XXFG, and XLFG was developed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) before and after digestion with purified isoprimeverose-producing oligoxyloglucan hydrolase (IPase). Using this method, the compositions of oligosaccharide units of soybean and mung bean xyloglucans were re-examined. Significant amounts of oligosaccharides that have not previously been reported to be oligosaccharide units of soybean and mung bean xyloglucans were found.
Pugc is a unique glutathione S-transferase subunit species that is absent in normal pumpkin plants, but GST3c (homodimer of Pugc) accumulates in cultured pumpkin cells treated with 40 ppm 2,4-dichlorophenoxyacetic acid for six days. GST3c was purified by DEAE-cellulose, hydroxylapatite, and S-hexylglutathione-agarose column chromatography, and its homogeneity was confirmed by SDS-PAGE analysis. The specific activity of GST3c was 124 μmol/min/mg protein with 1-chloro-2,4-dinitrobenzene. This was higher than those of the other plant and animal glutathione S-transferases. Mouse antiserum raised against the purified GST3c recognized only Pugc, but not Puga, Pugb, nor Pugd when the reactivity of pumpkin glutathione S-transferase subunits was tested.
We examined the effects of a peptidyl growth factor, phytosulfokine-α (PSK-α), isolated from conditioned medium and known to induce the proliferation of single mesophyll cells of asparagus, and its derivatives on chlorophyll formation in etiolated cotyledons of cucumber after illumination. The chlorophyll content was increased by PSK-α treatment. Chlorophyll-content increases were not observed following either [2-5]PSK or Tyr-SO3H treatment. There was no difference between the PSK-α treatments and water controls on increasing rate of fresh weights of cotyledons. These results suggest that PSK-α has the effect of elevating the chlorophyll content in the cells.
The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.
Sequences of 118 mRNAs of the green alga Chlamydomonas reinhardtii in the GenBank data base were compiled to examine the consensus sequence surrounding the AUG translation initiation codon. The consensus sequence for C. reinhardtii was found to be gc(A/C)A(A/C)(A/C)AUGGC. The AUG context of chloroplast proteins (nuclear coded) and non-chloroplast proteins were compared by a separate compilation, and some distinctive features in AUG context of chloroplast proteins were found.
We investigated the yield, gelation and sugar composition of water-soluble polysaccharides that had been extracted from soybean cotyledons under acidic conditions. SSPS were easily extracted at several pHs. The highest yield was about twice that reported by Morita et al., after extraction in boiling water, or by Kawamura et al., after extraction in hot water (90°C) with the stepwise addition of ammonium oxalate and 0.5% NaOH. Our results show that the average molecular weight was relatively high for both SSPS extracted in the weakly acidic pH range of 4-6 at 100-120°C and in the strongly acidic pH range of 2-3 at 40-80°C. The water-soluble polysaccharide solutions which were extracted under the former conditions remained fluid, but those which were extracted under the latter conditions gelled. However, the molecular weight distribution and sugar composition were similar for SSPS isolated by using both sets of conditions. Furthermore, water-soluble soybean polysaccharides were found to consist of rhamnogalacturonans, which could endure hydrolysis, and arabinogalactans, which were easily hydrolyzed under acidic conditions.
The effects of arginine on cell proliferation and subsequent T helper (Th) 1 and Th 2 cytokine synthesis by murine Peyer’s patch (PP) Th cells in vitro and the influence of arginine on the induction of antigen-specific mucosal and systemic immune responses in vivo were examined. When the PP T cells were stimulated with the anti-αβ T cell receptor (TCR) antibody in the presence of different concentrations of arginine, a higher proliferative response was observed in the culture with an optimal concentration of arginine compared with that with a minimum amount of this amino acid. The concentration of cytokines in the supernatant, the number of cytokine-producing cells and the cytokine-specific mRNA expression of PP T cells were also increased in a dose-dependent fashion. Furthermore, when mice fed on an arginine-supplemented liquid diet were orally immunized with tetanus toxoid plus cholera toxin as a mucosal adjuvant, a higher level of antigen-specific fecal IgA was observed when compared with the response in mice fed on an arginine-free diet. Taken together, these results suggest that arginine enhanced antigen-specific mucosal immune response resulting from the supporting activation of cell proliferation and subsequent cytokine synthesis of PP Th cells.
The effects of a D-methionine-containing solution (DMCS) on the nutritional status of AH109A hepatoma-bearing rats receiving total parenteral nutrition were studied. The DMCS solution inhibited the decrease of transferrin in the plasma of tumor-bearing rats when compared with the effect of an L-methionine-containing solution. The survival time was also significantly prolonged in the DMCS-treated rats. These results indicate that DMCS had a beneficial effect on the malnutrition induced in tumor-bearing rats and would be a useful amino acid solution for the nutritional support of cancer patients.
Gassericin A, a bacteriocin that was produced by Lactobacillus gasseri LA39, was treated with lysylendopeptidase and 3-bromo-3-methyl-2-(2-nitrophenyl-mercapto)-3H-indole. The fragments were recovered by SDS-PAGE and sequenced. All amino acids of gassericin A were distributed by sequence analysis and the bacteriocin did not contain any modified amino acids. The amino acid sequence of gassericin A completely coincided with that found through the cloning of the structural gene. Gassericin A was shown to be a cyclic bacteriocin (class II) which is bound at the N- and C-terminal ends. This is the first example of a cyclic bacteriocin from lactobacilli lactic acid bacteria.
We investigated the endurance swimming capacity of mice injected with CAP antagonist (capsazepine). The increase of endurance swimming capacity by the administration of CAP was significantly suppressed by the injection of capsazepine. At the same time, serum adrenaline secretion, which was induced by CAP, was depressed by capsazepine. These findings suggested that the increase in endurance swimming capacity by CAP was mediated by the CAP receptor.
Four major cis-lutein isomers could be identified after thermal isomerization of all-trans-lutein in a benzene solution system; namely, 13′-, 13-, 9′- and 9-cis-luteins. Using both all-trans-lutein and a mixture of these purified cis-luteins as starting materials, the quantitative changes determined by HPLC response show that this thermal isomerization was a reversible reaction. The equilibrium constants (kC) of 13′- and 13-cis-luteins were higher than those of 9′-, and 9-cis-luteins, suggesting that the amount formed of the former isomers was higher than that of the latter.
A novel phosphate ester containing a chromanol structure was synthesized from 1,2-diacyl-sn-glycero-3- phospho-2′-hydroxyethyl-2′,5′,7′,8′-tetramethyl-6′-hydro-xychroman (PCh) by hydrolysis catalyzed by phospholipase C from Bacillus cereus. The structure of the product was found by spectral analyses to be 2-(2′,5′,7′,8′-tetramethyl-6′-hydroxychromanyl)ethylphosphate (Ch-P). Ch-P was highly soluble in the aqueous phase at neutral pH values and exerted higher antioxidative activity than α-tocopherol and PCh in the Fe(III)/ascorbic acid-catalyzed peroxidation of a fish oil emulsion and the autoxidation of a rat brain homogenate.
A physical method is proposed that uses a cooling plate with many small holes to prevent initial supercooling in progressive freeze-concentration, and thus avoid serious contamination of the ice produced. The higher chance for ice nucleation of the water molecules in the holes due to the temperature gradient in the cooling plate resulted in the initial supercooling being completely prevented. Accordingly, the purity of the ice initially formed was substantially improved when compared with that by the standard vessel without holes in the cooling plate.
Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-β-galactosidases (P-β-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-β-gal I and II were cloned and sequenced. The structural gene of P-β-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-β-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-β-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-β-gal I and II with those of P-β-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-β-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no β-galactosidase activity, and that purified P-β-gal I and II can obviously hydrolyze o-nitrophenyl-β-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-β-gal I and II were each confirmed as a novel P-β-gal enzyme.
Bacteriocin ISK-1 is a proteinaceous inhibitory substance produced by Pediococcus sp. ISK-1 isolated from well-aged Nukadoko. Bacteriocin ISK-1 was purified by acid treatment, ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase HPLC from the culture supernatant of Pediococcus sp. ISK-1. Purification of bacteriocin ISK-1 resulted in a 30-fold increase in the specific activity and the recovery was 17%. Molecular mass of bacteriocin ISK-1 measured by fast atom bombardment-mass spectrometry was 2,960. The amino acid composition analysis of bacteriocin ISK-1 showed that it contained unusual amino acids such as lanthionine and/or 3-methyllanthionine, which is a characteristic of lantibiotics. The N-terminal amino acid sequence analysis indicated the first seven N-terminal amino acid residues as NH2-K-K-K-S-G-V-I. The primary sequence showed significant similarity to the lantibiotics lacticin 481 from Lactococcus lactis and variacin from Micrococcus varians, which suggests that bacteriocin ISK-1 is a novel lantibiotic belonging to a lacticin-481 type.
Enterococcus hirae has two sodium extrusion systems: the NapA Na+/H+ antiporter and the vacuolar Na+-ATPase. We found that a NapA mutant, WD4, which is deficient in Na+/H+ antiporter activity, grew well in the pH range of 6 to 10 up to 200 mM sodium. This was due to active, potential-independent sodium extrusion by the Na+-ATPase, which was induced under these conditions. The NapA Na+/H+ antiporter is thus not a prerequisite for growth of E. hirae in the presence of sodium, but plays a supplementary role in sodium extrusion at acidic pH.
Genomic DNA encoding Lipase I was cloned from Rhizopus niveus strain IFO4759. For expression of this gene in S. cerevisiae, DBY746 was transformed with YEp352PLipS, which had the cloned lipase gene under the control of a PGK promoter. This strain secreted the lipase at a high level (350 U/ml). The strain ND-12B was produced by a mating of DBY746, harboring YEp352PLipS, and NA74-3A, and dissection of asci. This new strain secreted the lipase up to 530 U/ml. Moreover, the lipase was produced most effectively in a medium containing Bacto-yeast extract, soy-peptide, and sucrose.
An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.